A New Species of the Genus Achalinus (Squamata: Xenodermidae) from the Dabie Mountains, Anhui, China

Simple Summary A new species of odd-scaled snake in the genus Achalinus is described from Dabie Mountains Luan City, Anhui Province, China, based on one male and two female specimens. Bayesian inference and maximum likelihood analyses based on a mitochondrial DNA fragment (CO1) indicated the new taxon is different from its congeners (p–distance ≥ 9.4%). Morphologically, the new species can be diagnosed from the other species by a combination of 12 characters. The recognition of the new species brings the number of described Achalinus species to 22. Abstract A new species of Xenodermid snake, Achalinus dabieshanensis sp. nov., was described based on three specimens (two female and one male) collected from the Dabie Mountains of western Anhui Province. It can be distinguished from known congeners by a significant genetic divergence in the mitochondrial gene fragment COI (p-distance ≥ 9.4%) and the following combination of characteristics: (1) length of the suture between the internasals being distinctly shorter than between the prefrontals; (2) a single loreal; (3) dorsal scales strongly keeled, in 23 rows throughout the body; (4) two pairs of prefrontals; (5) six supralabials; (6) five infralabials; (7) temporals 2 + 2 + 3 (or 2 + 2 + 4); (8) 141–155 ventrals; (9) 45–55 subcaudals, unpaired; (10) anal entire; (11) weakly iridescent tinged, uniform, brown to black dorsum with vertebral scales and about three adjacent dorsal scales dark brown forming a longitudinal vertebral line from posterior margin of parietals to tail tip; (12) light brown venter, ventral shields wide, visible on both sides, light brown flanks, giving the appearance of a black subcaudal streak. The recognition of the new species increases the number of described Achalinus species to 22.

During recent herpetological surveys of the Dabie Mountains, we collected three oddscaled snake specimens that differed from all other species of known snakes in their morphology and color pattern characteristics. According to the diagnostic characteristics of the genus Achalinus, as indicated by Smith and Zhao et al. [18,19], the specimens could be assigned to this genus. Further morphological examinations and molecular analyses showed that these specimens represent a separate evolutionary lineage that can be distinguished from all recognized species. Herein, we describe these specimens as a new species of the genus Achalinus.

Sampling
We obtained three odd-scaled snake samples from the Yaoluoping Nature Reserve and Fuziling Provincial Reserve in the Dabie Mountains, Anhui Province, China. The specimens were collected in the field and fixed in 75% ethanol. Liver tissue samples were separately transferred to 95% ethanol. The specimens have been deposited in the Biological Museum of Anhui University, Anhui, China. Permission for the field surveys in the Dabie Mountains was granted by the Huoshan Forestry Bureau (No. HFB20180801) and Management Office of the Yaoluoping National Reserve (No. YNR 20190503).

DNA Sequencing
Tissue samples from three individuals of the new specimens were used for the phylogenetic analyses. DNA was extracted from the liver samples using phenol/chloroform extraction [23]. The partial mitochondrial gene fragment of the cytochrome c oxidase 1 gene (COI) was amplified using the primer pair RepCOIF (5 ′ -TNT TMT CAA I ACC ACA AAG A-3 ′ ) and RepCOIR (5 ′ -ACT TCT GGR TGK CCA AAR AAT CA-3 ′ ) [24]. Experimental primer pairs were synthesized and provided by General Biosystems (Anhui) Co., Ltd. (Chuzhou, China). The PCR conditions followed those described by Nagy et al. (2012) [24]. The PCR products were sequenced by General Biosystems (Anhui) Co., Ltd. The sequences were edited and assembled using SeqMan (DNASTAR, Lasergene v7.1) and then aligned using Clustal X 1.8 [25] in MEGA6 [26]. Sequences with insertions or deletions (indels) were sequenced at least twice with both the forward and reverse primers to confirm the variations. All sequences were deposited in GenBank (Table 1).

Phylogenetic Analyses
To explore the phylogenetic position of the samples in the genus Achalinus, COI sequences from 21 snakes, including three outgroups, Fimbrios klossi, Parafimbrios lao, and P. vietnamensis, were retrieved from GenBank for our analyses (Accession number listed in Table 1) [16,17]. The nucleotide substitution models were tested in MrModeltest 2.3 using Akaike information criteria [27], and the GTR + G + I model was found to be the best fit. Bayesian analyses were conducted using MrBayes 3.2.4 [28]. Three independent runs with four Markov Chain Monte Carlo were performed based on the models selected, starting from a random tree; each run consisted of a total of 3 × 10 6 generations, sampled every 2000 generations. The first 25% of the samples were discarded as burn-in. The remaining trees were used to construct a 50% majority rule consensus tree. The re-sults were analyzed in Tracer v1.7 to assess convergence and ensure effective sample sizes (≥200) for all parameters [29]. Maximum likelihood analyses were executed using IQ- TREE 2 (Minh et al., 2020), and the support of the tree was by bootstrap resampling with 1000 nonparametric bootstrap replicates. Phylogenetic trees were visualized and edited using FigTree v1.4.2 [30]. We also calculated the uncorrected pairwise genetic distances (p-distance) using MEGA 6 [25].

Morphometric Measurements
Morphometric measurements to the nearest 0.1 mm were taken using dial calipers, following Zhao et al. (1998) [19]. The abbreviations of morphological characteristics used in the text are as follows: total length (TL, from snout tip to tail end); snout-vent length (SVL, from cusp of snout to anterior margin of cloacae opening); tail length (TaL, from posterior margin of cloacae opening to tip of tail); head length (HL, from snout tip to posterior margin of mandible); head width (HW, at the widest part of the head); head height (HH, at the highest part of the head); length of loreal (LeL, at the longest part of the loreal); height of loreal (HiL, at the highest part of the loreal); length of anterior section of nasal (LaSN); length of posterior section of nasal (LpSN); length of suture between prefrontals (LSBP); length of suture between internasals (LSBI); preoculars (PrO); postoculars (PtO); supraoculars (SpO); supralabials (SPL); infralabials (IFL); anterior temporals (aTMP); middle temporals (mTMP); posterior temporals (pTMP); ventral scales (V); and subcaudals (SC). Dorsal scale rows (DSR) were counted at two head lengths behind the head, mid-body, and two head lengths before the vent. Bilateral scale counts are given as left/right. Sex identification was performed by inspecting the presence or absence of hemipenes. In this study, while comparing the undescribed species with A. rufescens, the most widespread species, we used its latest description of A. rufescens rather than descriptions in the early literature, because it was very likely that multiple cryptic species will be involved in them [10]. In the morphometric analysis, we focused on the species that clustered most closely with the new specimen in the phylogenetic trees. Morphological data for the Achalinus species were obtained from the relevant literature sources [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17].
The electronic version of this articIe in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information can be viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/ (accessed on 22 October 2022). The LSID for this publication is urn:lsid:zoobank.org:pub: 1E1842EA-CB20-46FA-AA0D-3D35017814D4.

Phylogenetic Relationship
The alignment contained 657 nucleotide base pairs (bp) sequences. The Bayesian inference (BI) and Maximum likelihood (ML) phylogenetic results ( Figure 1) were largely concordant concerning the relationships among species within the major clades of Achalinus. The Achalinus lineages formed a monophyletic group with high support values (BI, PP = 1.00; ML, BS = 100%). These three specimens from the Dabie mountain were nested within the Achalinus genus and the independence of our collections and their affinity to A. huangjietangi were also strongly supported (BI, PP = 1.00; ML, BS = 82%). In addition, these new samples have distinct branch lengths compared with the adjacent clade of A. huangjietangi (HSR18030 collection number).
The uncorrected p-distance among all species within the genus Achalinus ranged from 6.1% to 21.7% ( Table 2). The maximum genetic distance was found between the new species and A. formosanus (21.7%), and the minimum one was found between the new species and A. huangjietangi (9.4%) ( Table 2). The genetic distance between the new samples and A. huangjietangi (9.4%) is greater than the lowest three ones: (7.5% between A. ater and A. juliani, 6.5% between A. yangdatongi and A. juliani, and 6.1% between A. ningshanensis and A. yangdatongi).
Given that these specimens possess obvious monophyletic structures in the molecular phylogenetic tree, adding the significant molecular distance, unique geographical distribution, and prominent morphological differences from congenetic species, they are described as a new species below.   Table 1.

Comparisons
Achalinus dabieshanensis sp. nov. differs from all other species of Achalinus, except A. formosanus, A. huangjietangi, A. niger, and A. spinalis by having internasal suture lengths distinctly shorter than the lengths of the suture between the prefrontals (vs. other species of Achalinus, length of suture between the internasals distinctly longer than or equal to that between the prefrontals). In addition, it differs from all other species of Achalinus, except A. dehuaensis, A. emilyae, A. hainanus, A. huangjietangi, A. rufescens, A. meiguensis, Table 4.
Nostril in anterior part of nasal, posterior margin of nostril with distinct nostril cleft, posterior section of nasal vertically rectangular, posterior section nearly half as long as anterior section, LaSN/LpSN 0.4; single pentagonal frontal, nearly straight anteriorly, slightly broader than long, pointed backwards, much shorter than parietals; single loreal, HiL/LeL ratio 0.8, extending from nasal to eye; single supraocular, elongated, twice as wide as high; two anterior temporals, elongated, upper one smaller, widely in contact with eye, lower one narrowly in contact with eye; two elongated middle temporals, upper one much larger, lower one in contact with 6th supralabial, not in contact with elongated anterior temporals on left, only tip in contact with anterior temporals on right; three elongated posterior temporals, uppermost one significantly enlarged (super-temporal), surrounding the parietal; each parietal bordered by elongated nuchal; nuchals separated from each other behind super-temporals by one small intertemporal nuchal scale; 2nd nuchal about twice size of 1st; six supralabials, 1st smallest, 4th and 5th widely in contact with eye, 6th longest and largest; 3rd and 4th in broad contact with loreal; one mental, followed by five infralabials with first pair in contact with each other; two pairs anterior and posterior chin shields in contact with 3rd infralabial; posterior pair of chin shields smaller, length of suture between 1st pair twice that between 2nd pair; dorsal scales lanceolate and feebly keeled; dorsal scales in 23 rows throughout body, outermost rows on both sides smooth and significantly enlarged; 155 ventrals, distinctly rounded laterally; 45 subcaudals, not paired; anal entire.
The coloration of the holotype in life Dorsal surface is uniform iridescent brown, with a longitudinal dark brown vertebral line, a width of about 3 DSR, from posterior margin of parietals to tail tip, ventrals light brown; margins of all scales grayish white; coloration of supralabials and temporal regions much lighter; iris dark brown, pupil black.

Intraspecific Morphological Variations
The measurements, scale counts, body proportions, and squamae details are listed in Table 3. All paratypes are morphologically very similar to the holotype except that:

Etymology
The specific epithet, Achalinus dabieshanensis sp. nov., refers to the distribution of the new species in the Dabie Mountains in Anhui, China. We suggest the English name would be the "Dabie Mountains Odd-scaled Snake" or "Dabie Mountain Burrowing Snake" and the Chinese name " 大别山脊蛇 (Dà Bié Shān Jĭ Shé)".

Distribution and Habitat
Currently, Achalinus dabieshanensis sp. nov. is only known from its type-locality, Fuziling Provision Reserve, Yaoluoping Nature Reserve, and an adjacent area in the Dabie Mountains, Anhui, China ( Figure 5). The new species was discovered in the leaf litter of a well-preserved montane evergreen deciduous broad-leaved mixed forest (635-1361 m a.s.l.).

Discussion
Achalinus is an ancient group, which diverged from its closest related genus approximately 77.4 million years ago, and is widely distributed in China, Vietnam, and Japan now [4,18,19,31]. However, the morphological characteristics of the genus Achalinus are relatively conservative, such as its color, number of scales, etc. [20], which require detailed morphological differences comparison and more molecular evidence to distinguish species [14,20]. Thus, for Achalinus, slight morphological differences often play key roles in species delimitation. For example, in 2019, A. yunkaiensis was discovered in Guangdong; it only differs from A. spinalis by comparing the length of suture between the internasals and that between the prefrontals [10]. Another example is that A. huangjietangi only differs from A. yunkaiensis by a dark streak in the middle of the caudal ventral [14]. In addition, A. huangjietangi differs from A. spinalis by the number of anterior temporals in contact with the eye and a dark streak in the middle of the caudal ventral [14]. In contrast, there are often very substantial genetic differentiations between these species. Given the significant molecular distance within the specimens of A. huangjietangi, we suspect that there are one or more cryptic species to be further investigated; because of the lack of the morphological data, we could not further investigate the taxonomic relationship within those specimens [14]. Temporarily in this study, they are labeled as sp. 1 and sp. 2 ( Figure 1).
In this study, ML and BI trees showed that A. dabieshanensis sp. nov. is the sister to A. huangjietangi (Figure 1), and these two lineages have distinct morphologies (Figure 3, Table 4). Moreover, the difference between A. dabieshanensis and A. huangjietangi is also reflected in their significant genetic divergences (p-distance = 9.4%, Table 2); their genetic distance is higher than that among many species in the genus. In general, Achalinus species tend to inhabit wetter, more mountainous areas with low dispersal ability [9], so large

Discussion
Achalinus is an ancient group, which diverged from its closest related genus approximately 77.4 million years ago, and is widely distributed in China, Vietnam, and Japan now [4,18,19,31]. However, the morphological characteristics of the genus Achalinus are relatively conservative, such as its color, number of scales, etc. [20], which require detailed morphological differences comparison and more molecular evidence to distinguish species [14,20]. Thus, for Achalinus, slight morphological differences often play key roles in species delimitation. For example, in 2019, A. yunkaiensis was discovered in Guangdong; it only differs from A. spinalis by comparing the length of suture between the internasals and that between the prefrontals [10]. Another example is that A. huangjietangi only differs from A. yunkaiensis by a dark streak in the middle of the caudal ventral [14]. In addition, A. huangjietangi differs from A. spinalis by the number of anterior temporals in contact with the eye and a dark streak in the middle of the caudal ventral [14]. In contrast, there are often very substantial genetic differentiations between these species. Given the significant molecular distance within the specimens of A. huangjietangi, we suspect that there are one or more cryptic species to be further investigated; because of the lack of the morphological data, we could not further investigate the taxonomic relationship within those specimens [14]. Temporarily in this study, they are labeled as sp. 1 and sp. 2 ( Figure 1).
In this study, ML and BI trees showed that A. dabieshanensis sp. nov. is the sister to A. huangjietangi (Figure 1), and these two lineages have distinct morphologies ( Figure 3, Table 4). Moreover, the difference between A. dabieshanensis and A. huangjietangi is also reflected in their significant genetic divergences (p-distance = 9.4%, Table 2); their genetic distance is higher than that among many species in the genus. In general, Achalinus species tend to inhabit wetter, more mountainous areas with low dispersal ability [9], so large rivers may form insurmountable geographical barriers [13]. In this study, the Yangtze River acted as the geographical barrier separating A. huangjietangi from the A. dabieshanensis sp. nov.. Similar situations have been reported in adjacent species, such as A. timi and A. zugorum; they were separated by the Red River [13]. Therefore, the limited dispersal ability and geographical barriers may have probably led to species divergence between A. huangjietangi and A. dabieshanensis sp. nov. Based on the molecular and morphological evidence, we think that A. dabieshanensis sp. nov. should be considered a valid species.
The discovery of A. dabieshanensis sp. nov. extends the distribution of the genus northward to Dabie Mountain in the lower reaches of the Yangtze River. To date, many new vertebrate species have been discovered in the Dabie Mountains [32][33][34][35][36][37]; the discovery of the A. dabieshanensis sp. nov. further suggests that species diversity in the Dabie Mountains remains underestimated to some extent. At present, based on the collection sites of the three new specimens, it is speculated that A. dabieshanensis sp. nov. may be distributed throughout the Dabie Mountains. Given that the secretive nature of odd-scaled snakes makes their discovery largely serendipitous [10], we cannot make exact judgments about the abundance and population status of the new species in the Dabie Mountain area. Therefore, it is difficult to assess the risk of extinction of this species for the time being. We recommend classifying A. dabieshanensis sp. nov. as Data Deficient (DD) on the IUCN Red List. It is worth noting that human activities in the Dabie Mountains in recent years, including vegetation destruction, road construction, and artificial surface expansion [38], may threaten this species.

Conclusions
A new species of Achalinus, Achalinus dabieshanensis sp. nov., is described based on three specimens collected from the Dabie Mountains of western Anhui Province. It appears to be widespread in the Dabie Mountains. The discovery of new species made the members of the genus Achalinus distribution area extend northward to the Dabie Mountain in the lower reaches of the Yangtze River. However, their discovery is largely accidental, which makes it difficult for us to make accurate judgments on the abundance and population status of this new species in the Dabie Mountains. Further investigations will be necessary to assess the risk of extinction of this species.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.